Somatostatin analog and process for its preparation

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molecule from undergoing reaction during subjection of the molecule to conditions which could cause disruption of the otherwise active moiety. Secondly, the protecting group is such as can be readily removed with restoration of the original active moiety and under conditions which would not undesirably affect other portions of the molecule. Groups which are useful for these purposes, that is, for protecting amino, hydroxy, and thio groups, are well recognized by those skilled in the art. Indeed, entire

1q volumes have been directed specifically to a description and discussion of methods for using such groups. One such volume is the treatise Protective Groups in Organic Chemistry, J.F.W. McOmie, Editor, Plenum Press, New York, 1973.

starting compounds In the above formulas defining the intormediate«, conventional R represents either an a-amino hydrogen or a#/a-amino protecting group. The a-amino protecting. groups contemplated for R are well recognized by those of ordinary skill in the peptide art. Many of these are detailed in McOmie, supra, Chapter 2, authored by J. W. Barton. Illustrative of such protecting groups are benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, g-bromobenzyloxycarbony1, o-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, o-bromobenzyloxycarbonyl, g-methoxybenzyloxycarbonyl, g-nitrobenzyloxycarbonyl, t-butyloxycarbonyl (BOC), t-amyloxycarbonyl, 2-(g-biphenylyl) isopropyloxycarbonyl (BpOC) , adamantyloxycarbonyl, isopropyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl, trLpheny!methyl (trityl), and g-toluenesulfonyl. Preferably, the a-amino protecting group defined by R is t-butyloxycarbonyl.

X-4747A -6־ ׳':. eV represents either the hydrogen of the sulfconventional hydryl group o! the cysteine or a/protecting group for the sulfhydryl substituent. Many such protecting groups are described in M&lt;:Omie, supra, Chapter 7, authored by R, G. Hickey, V. R. Rao, and W, G. Rhodes. Illustrative suitable such protectintf groups are g-methoxybenzyl, benzyl, £tolyl, benzhydryl, acetamidomethyl, trityl, g-nitrobenzyl, t-butyl, isobutyloxymethyl, as well as any of a number of trityl derivatives. For additional groups, see, for example,

Houben-Weyl, Methodes der Organischen Chemie, Synthese von

Peptiden, Vols. 15/1 and 15/2, (1974), Stuttgart, Germany.

Preferably, the sulfhydryl protecting group defined by R is g-methoxybenzy).

R2 represents either hydrogen on the c-amino conventional function of the lysine residue or a suitable/ε-amino protecting group. Illustrative of such groups are the bulk of those mentioned hereinabove as being suitable for use as an a-amino protecting group. Included as typical such groups are benzyloxycarbonyl, t-butyloxycarbony1, t-amyloxycar20 bonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, pmethoxybenzylox/carbonyl, p-chlorobenzyloxycarbonyl, pbromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,6dichlorobenzylo.xycarbonyl, 2,4-dichlorobenzyloxycarbonyl, o-bromobenzylox/carbony1, ρ-nitrobenzyloxycarbonyl, isopropyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl, and p--toluenesulfonyl.

As will become apparent hereinafter, the process for the preparat ion of the tetradecapeptides of formula I

X-4747A involves periodic cleavage of the a-amino protecting group from the terminal amino acid present on the peptide chain. Thus, the only limitation with respect to the identity of the ε-amino protecting group on the lysine residue is that it be such that it will not be cleaved under the conditions employed to selectively cleave the a-amino protecting group. Appropriate selection of the a-amino and the ε-amino protecting groups is a matter well within the knowledge of a peptide chemist of ordinary skill in the art 10 and depends upon the relative ease with which a particular protecting group can be cleaved. Thus, groups such as 2-(p-biphenylyi)isopropyloxycarbonyl (BpOC) and trityl are very labile and can be cleaved even in the presence of mild acid. A moderately strong acid, such as hydrochloric acid, trifluoroacetic acid, or boron trifluoride in acetic acid, is required to cleave other groups such as t-butyloxycarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl, and £methoxybenzyloxycarbonyl. Even stronger acid conditions are required to effect cleavage of other protecting groups such 20 as benzyloxycarbonyl, halobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl, and isopropyloxycarbonyl. Cleavage of these latter groups requires drastic acid conditions such as the use of hydrogen bromide, hydrogen fluoride, or boron trifluoroacetate in trifluoroacetic acid. Of course, any of the more labile groups will also be cleaved under the stronger acid conditions. Appropriate selection of the amino protecting groups thus will include the use of a group at the a-amino function which is more labile than that employed as the ε-amino protecting group 30

X-4747A coupled with cleavage conditions designed to selectively remove only the a-amino function. In this context, R₂preferably is o-chlorobenzyloxycarbonyl or cyclopentyloxycarbonyl, and, in conjunction therewith, the a-amino protecting group of choice for use in eachof the amino acids which is added to the peptide chain preferably is t-butyloxycarbonyl.

The groups R^ and R^ represent the hydroxyl conventional hydrogen or a/protecting group for the alcoholic hydroxyl of threonine and serine, respectively. Many such protecting groups are described in McOmie, supra, Chapter 3, authored by C. B. Reese. Typical such protecting groups are, for example, ryc^ alkyl, such as methyl, ethyl, and t-butyl; benzyl; substituted benzyl, such as [)-methoxybenzyl, £nitrobenzyl, [)-chlorobenzyl, and o-chlorobenzyl; C₁־C₃alkanoyl, such as formyl, acetyl, and propionyl; triphenyl methyl (trityl). Preferably, when. R^ and are protecting groups, the protecting group of choice in both instances is benzyl.

The group R₅.represents either hydrogen or formyl and defines the moiety yiRp of the tryptophan residue. The formyl serves as a protecting group. The use of such a protecting group is optional and, therefore, R^ properly can be hydrogen (N-anprotected) or formyl (N-protected).

The group X relates to the carboxyl terminal of the tetradecapeptide chain; it can be hydroxyl, in which case a free carboxyl group is defined. In addition, X represents the solid resin support to which the carboxyl

X-4747A terminal moiety of the peptide is linked during its synthesis. This solid resin is represented by the formula

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In any of the above, when X represents hydroxyl, each of R, , , R^, , and is hydrogen. When X represents the solid resin support, each of R, R,, R_ , R,_׳12 3 and is a protecting group.

The following abbreviations, most of which are well known and commonly used in the art, are employed herein:

Ala - Alanine

Asn - Asparagine

Cys - Cysteine

Gly - Glycine

Lys ־ Lysine

Phe ־ Phenylalanine

Ser - Serine

Thr - Threonine

Trp - Tryptophan

Vai - Valine

DCC - N,N¹-Dicyclohexylcarbodiimide

DMF - N,N-Dimethylformamide

BOC - t-Butyloxycarbonyl

PMB - p-Methoxybenzyl

CBzOC - o-Chlorobenzyloxycarbonyl

CPOC - Cyclopentyloxycarbonyl

Bzl - Benzyl

X-4747A

For - Formyl

BpOC - 2-(g-biphenylyl)isopropyloxycarbonyl

Although the selection of the particular protecting groups to be employed in preparing the compounds of formula I remains a matter well within the ordinary skill of a synthetic peptide chemist, it is well to recognize that the sequence oi reactions which must be carried out gives rise to a selection of particular protecting groups. In other words, the protecting group of choice must be one 10 which is stable both to the reagents and under the conditions employed in the succeeding steps of the reaction sequence. For example, as already discussed to some degree hereinabove, the particular protecting group which is employed must be one which remains intact under the conditions which are employed for cleaving the a-amino protecting group of the terminal amino acid residue of the peptide fragment in preparation for the coupling of the next succeeding amino acid fragment to the peptide chain. It is also important to select, as a protecting group, one which 20 will remain intact during the building of the peptide chain and which will be readily removable upon completion of the synthesis of the desired tetradecapeptide product. All of these matters are well within the knowledge and understanding of a peptide chemist of ordinary skill in the art.

As is evident from the above discussion, the tetradecapeptides of formula I can be prepared by solid phase synthesis. This synthesis involves a sequential building of the peptide chain beginning at the C-terminal 30

X-4747A end of the peptide. Specifically, cysteine first is linked at its carboxyl function to the resin by reaction of an amino-protected, S-protected cysteine with a chloromethyl־ ated resin or a hydroxymethyl resin. Preparation of a hydroxymethyl resin is described by Bodanszky et al., Chem. Ind. (London), 38 1597-98 (1966). The chloromethylated resin is commercially available from Lab Systems, Inc., San Mateo, California.

In accomplishing linkage of the C-terminal cys10 teine to the resin, the protected cysteine first is converted to its cesium salt. This salt then is reacted with the resin in accordance with the method described by B.F. Gisin, Helv. Chim. Acta, 56, 1476 ¢1973) . Alternatively, the cysteine can be linked to the resin by activation of the carboxyl function of the cysteine molecule by application of readily recognized techniques. For example, the cysteine can be reacted with the resin in the presence of a carboxyl group activating compound such as N,N'-dicyclohexylcarbodiimide (DCC).

Once the free carboxyl cysteine has been appropriately linked to the resin support, the remainder of the peptide building sequence involves the step-wise addition of each amino acid to the N-terminal portion of the peptide chain. Necessarily, therefore, the particular sequence which is involvad comprises a cleavage of the a-amino protecting grouo from the amino acid which represents the N-terminal portion of the peptide fragment followed by coupling of the next succeeding amino acid residue to the

X-4747A now free and reactive N-terminal amino acid. Cleavage of the a-amino protecting group can be effected in the presence of an acid such as hydrobromic acid, hydrochloric acid, trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and acetic acid, with formation of the respective acid addition salt product. Another method which is available for accomplishing cleavage of the amino protecting group involves the use of boron trifluoride. For example, boron trifluoride diethyl etherate in glacial acetic acid will convert the amino-protected peptide fragment to a BF^ complex which then can be converted to the deblocked peptide fragment by treatment with a base such as aqueous potassium bicarbonate. Any of these methods can be employed as long as it is recognized that the method of choice must be one which accomplishes cleavage of the N-terminal a-amino protecting group without disruption of any other protecting groups present on the peptide chain. In this regard, it is preferred that the cleavage of the N-terminal protecting group be accomplished using trifluoroacetic acid. Generally, the cleavage will be carried out at a temperature from about 0°C. to about room temperature.

Once the N-terminal cleavage has been effected, the product which results normally will be in the form of the acid addition salt of the acid which has been employed to accomplish the cleavage of the protecting group. The product then can be converted to the free terminal amino compound by treatment with a mild base, typically a tertiary amine such as pyridine, or triethylamine.

X-4747A

The peptide chain then is ready for reaction with the next succeeding amino acid. This can be accomplished by employing any of several recognized techniques. In order to achieve coupling of the next-succeeding amino acid to the N-terminal peptide chain, an amino acid which has a free carboxyl but which is suitably protected at the a-amino function as well as at any other active moiety is employed. The amino acid then is subjected to conditions which will render the carboxyl function active to the coupling re10 action. One such activation technique which can be employed in the synthesis involves the conversion of the amino acid to a mixed anhydride. Thereby, the free carboxyl function of the amino acid is activated by reaction with another acid, typically a carbonic acid in the form of its acid chloride. Examples of such acid chlorides which can be used to form the appropriate mixed anhydrides are ethyl chloroformate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chloroformate, and pivaloyl chloride.

Another method of activating the carboxyl function 20 of the amino acid to achieve coupling is by conversion of the amino acid to its active ester derivative. Examples of such active esters are, for example, a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, a p-nitrophenyl ester, an ester formed from 1-hydroxybenzotriazole, and an ester formed from N-hydroxysuccinimide. Another method for effecting coupling of the C-terminal amino acid to the peptide fragment involves carrying out the coupling reaction in the

X-4747A presence of at least an equimolar quantity of N,N'-dicyclohexylcarbodiimide (DCC). This latter method is preferred for preparing the tetradecapeptide of formula II where X is /־־y^{Re ח 1 כ}

-O-CH . ' ^s W

Once the desired amino acid sequence has been prepared, the resulting peptide can be removed from the resin support. This is accomplished by treatment of the protected resin-supported tetradecapeptide with hydrogen fluoride. Treatment with hydrogen fluoride cleaves the peptide from the resin; in addition, however, it cleaves all remaining protecting groups present on the reactive moieties located on the peptide chain as well as the a-amino protecting group present at N-terminal amino acid. When hydrogen fluoride is employed to effect the cleavage of the peptide from the resin as well as to remove the protecting groups, it is preferred that the reaction be carried out in the presence of anisole. The presence of anisole has been found to inhibit the potential alkylation of certain amino acid residues present in the peptide chain. In addition, it is preferred that the cleavage be carried out in the presence of ethyl mercaptan. The ethyl mercaptan serves to protect the indole ring of the tryptophan residue, and, furthermore, it facilitates conversion of the blocked cysteines to their thiol forms. Also, when R₅ is formyl, the presence of ethyl mercaptan facilitates hydrogen fluoride cleavage of the formyl group.

X-4747A

Once the cleavage reaction has been accomplished, the product which is obtained is a straight-chain peptide containing 14 amino acid residues. In order to obtain the final product of formula 1, it is necessary to treat the straight-chain tetradecapeptide under conditions which will effect its oxidation by converting the two sulfhydryl groups present in the molecule, one at each cysteinyl moiety, to a disulfide bridge. This can be accomplished by treating a dilute solution of the linear tetradecapeptide 10 with any of a variety of oxidizing agents including, for example, iodine, and potassium ferricyanide. Air also can be employed as oxidizing agent, the ph of the mixture generally being from about 2.5 to about 9.0, and preferably from about 6.2 to about 7.2. When air is used as oxidizing agent, the concentration of the peptide solution generally is not greater than about 0.4 mg. of the peptide per milliliter of solution, and usually is about 50 pg./ml.

The compounds of formula I may be administered to warm-blooded mammals, including humans, and are particularly 20 useful for relaxing smooth muscle. Specifically, the gastrointestinal tract can be relaxed by parenteral administration of small amounts of these compounds, and preferably, of 1 8

D-Val ,D-Trp -somatostatin. This action, resulting in reduction of gut motility, is particularly desirable in hypotonic gastrointestinal radiography. These compounds, furthermore, are useful in treatment of spastic colon, pylorospasm, and other spastic conditions of the gastrointestinal tract, as well as for ureteral and biliary colic.

X-4747A

Normally, in order to effect relaxation of smooth muscle, these compounds are administered at a dose of about 0.1 ng. to about 3 ug. per kilogram body weight of the recipient, and preferably from about 0.3 ng. to about 1.5 ng. per kilogram body weight. Administration is parenteral, and it can be intramuscular, subcutaneous, or intravenous; preferably, the compounds are administered intravenously or intramuscularly.

For parenteral administration, fluid unit dosage' 10 forms generally are prepared using the compound in association with a pharmaceutical carrier, such as, for example, isotonic saline, isotonic glycine, lactose, mannitol, dilute acetic acid, bacteriostatic water, for example, water containing about 1% benzyl alcohol, and phosphate buffer solutions, as well as appropriate combinations of any standard carrie׳rs. The carrier, relative to the active compound, generally is present in a weight ratio of from about 25:1 to about 1000:1.

The compound, depending upon the carrier and the 20 concentration used, can either be suspended or dissolved in a suitable sterile vehicle, water being preferred. In preparing solutions, the compound and carrier can be dissolved in the selected vehicle, the solution filtered and added to a suitable vial or ampoule, and the vial or ampoule sealed. Advantageously, adjuvants, such as a local anesthetic preservative or a buffering agent, can be dissolved in the vehicle. To enhance stability, the compound in association with the carrier can be dissolved in water, and

X-4747A the aqueous solution placed into a vial and then lyophilized. The dry lyophilized solid then is sealed in the vial and an accompanying vial of the vehicle supplied to reconstitute the composition prior to use. Parenteral suspensions can be prepared in substantially the same manner except that the compound is suspended in the carrier instead of being dissolved.

The compounds of formula I also are active, although not necessarily to an equivalent degree, in inhibiting the release of growth hormone. This inhibitory effect is 10 beneficial in those instances in which the host being treated requires a therapeutic treatment for excess secretion of somatotropin, such secretion being associated with adverse conditions such as juvenile diabetes and acromegaly. These compounds also exhibit other physiological effects, including the inhibition of gastric acid secretion, useful in treatment of ulcer conditions; the inhibition of exocrine pancreas secretion, potentially useful in treatment of pancreatitis; and the inhibition of secretion of insulin and glucagon. The compounds may be administered by any of 20 several methods, including oral, sublingual, subcutaneous, intramuscular, and intravenous. Preferably, the dose range for sublingual or oral administration is about 1 mg. to about 100 mg./kg. of body weight. Generally, the intravenous, subcutaneous, or intramuscular dose range for these latter indications is from about 1 pg. to about 1 mg./kg. of body weight, and, preferably, is from about 50 pg. to about 100 pg./kg. of body weight. It is evident that the dose range will vary widely depending upon the particular condition which is being treated as well as the severity of the 30 condition.

X-4747A

The compounds of formula .1 can be administered orally or sublingually in association with a pharmaceutical carrier, for example, in the form of tablets or capsules. Inert diluents or carriers, for example, magnesium carbonate or lactose, can be used together with conventional disintegrating agents, for example, maize starch and alginic acid, and lubricating agents, for example, magnesium stearate. Typically, the amount of carrier or diluent will range from about 5 to about 95 percent of the final composition, and 10 preferably from about 50 to about 85 percent of the final composition. Suitable flavoring agents also can be employed in the final preparation rendering the composition more palatable for administration.

When the compounds of formula I are to be administered parenterally, suitable carriers may be employed, such as, for example, any of those described above with reference to the use of these compounds for relaxing smooth muscle.

The following examples are illustrative of the preparation of compounds of formula I and intermediates 20 thereto.

Example 1

N-t-BUTYLOXYCARBONYL-L-CYSTElNYL (S-p-METHOXYBENZYL) METHYLATED POLYSTYRENE RESIN

To 1000 ml. of N,N-dimethylformamide (DMF) containing the cesium salt of N-t-butyloxycarbonyl-(S-pmethoxybenzyl)cysteine (prepared from 17.5 g. of the free acid) were added 100 g. of chloromethylated polystyrene resin (Lab Systems, Inc., 0.75 mmoles Cl/gram). The mixture was stirred at room temperature for five days. The resin

X-4747A then was filtered and was washed .alternately three times each with a mixture of 85 percent DMF and 15 percent water and with DMF, and then twice with DMF, To the resin suspended in 1000 ml. of DMF were added a solution of 16 grams (83.4 mmoles) of cesium acetate. The mixture was stirred for nine days at room temperature. The resin then was filtered and was washed alternately three times each with a mixture of 85 percent DMF and 15 percent water and with DMF. The resin was washed with CHCl^ and then was suspended four times in CHCl^ in a separatory funnel, drawing off the liquid each time to remove fines. The resin was filtered, washed with 95% ethanol and then alternately three times each with benzene and 95 percent ethanol. The resin then was dried in vacuo at 30^cC. to obtain 115.3 g. of the title product. An amino acid analysis showed 0.254 mmoles of Cys per gram resin. The cysteine was determined as cysteic acid from an acid hydrolysis carried out using a 1:1 mixture of dioxane and concentrated hydrochloric acid to which a small amount of dimethyl sulfoxide was added.

Example 2

N-t-BUTYLOXYCARBONYL-D-VALYL-GLYCYL-L-(S-p-METHOXYBENZYL)CYSTEINYL-L-(N׳ -o-CHLOROBENZYLOXYCARBONYL)LYSYL-L-ASPARAGINYL-L-PHENYLALANYL-L-PHENYLALANYLD-TRYPTOPHYL-L-(N -o-CHLOROBENZYLOXYCARBONYL)LYSYL-L(O-BENZYL)THREONYL-L-PHENYLALANYL-L-(O-BENZYL)THREONYLL-(O-BENZYL)SERYL-L-(S-p-METHOXYBENZYL)CYSTEINYL METHYLATED POLYSTYRENE RESIN

The product from Example 1 (5.0 grams) was placed !ת the reaction vessel of a Beckman 990 automatic peptide synthesizer, and twelve of the remaining thirteen amino acids were added employing the automatic synthesizer. The

X-4747A resulting protected tridecapeptide resin was divided into two equal portions, and the final residue was introduced to one of the portions. The amino acids which were employed as well as the sequence of their employment is as follows: (1) N-t-butyloxycarbonyl-(O-benzyl)-L-serine; (2) N-t-butyloxycarbonyl-(O-benzyl)-L-threonine; (3) N-t-butyloxycarbonylL-phenylalanine; (4) N-t-butyloxycarbonyl-(O-benzyl)-Lthreonine; (5) N^a-t-butyloxycarbonyl-N^£-o-chlorobenzyloxycarbonyl-L-lysine; (6) N^a-t-butyloxycarbonyl-D-tryptophan;

(7) N-t-butyloxycarbonyl-L-phenylalanine; (8) N-t-butyloxycarbonyl-L-phenylalanine; (9) N-t-buty.loxycarbonyl-Lasparagine, p-nitrophenyl ester; (10) N^a-t-butyloxycarbonyl£

N -o-chlorobenzyloxycarbonyl-L-lysine; (11) N-t-butyloxycarbonyl-(S-g-methoxybenzyl)-L-cysteine; (12) N-t-butyloxycarbonyl-glycine; and (13) N-t-butyloxycarbonyl-Dvaline. The sequence of deprotection, neutralization, coupling, and recoupling for the introduction of each amino acid into the peptide is as follows: (1) three washes (10 ml./gram resin) of three minutes each with chloroform; (2) 20 removal of BOC group by treatment twice for twenty minutes each with 10 ml./g.ram resin of a mixture of 29 percent trifluoroacetic acid, 48 percent chloroform, 6 percent triethylsilane, and 17 percent methylene chloride; (3) two washes (10 ml./gram resin) of three minutes each with chloroform; (4) one wash (10 ml./gram resin) of three minutes with methylene chloride; (5) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (6)

X-4747A -21three washes (10 ml./gram resin) of three minutes each with methylene chloride; (7) neutralization by three treatments of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (8) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (9) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (10) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (11) addition of 10 1.0 mmole/gram resin of the protected amino acid and 1.0 mmole/gram resin of N,N'-dicyclohexylcarbodiimide (DCC) in 10 ml./gram resin of methylene chloride followed by mixing for 120 minutes; (12) three washes (10 ml,/gram resin) of three minutes each with methylene chloride; (13) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (14) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (15) neutralization by three treatments of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (16) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (17) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (18). three washes (10 ml./ gram resin) of three minutes each with methylene chloride; (19) three washes (10 ml./gram resin) of three minutes each with DMF; (20) addition of 1.0 mmole/gram resin of the protected amino acid and 1.0 mmole/gram resin of N,N׳-dicyclohexylcarbodiimide (DCC) in 10 ml./gram resin of 30

X-4747A a 1:1 mixture of DMF and methylene chloride followed by mixing for 120 minutes; (21) three washes (10 ml./gram resin) of three minutes each with DMF; (22) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (23) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (24) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (25) neutralization by three treatments of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (26) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (27) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; and (28) three washes (10 ml./gram resin) of three minutes each with methylene chloride.

The above treatment sequence was employed for addition of each of the amino acids with the exception of the glycine and asparagine residues. The glycine addition 20 was carried out using only steps 1-18. The asparagine residue was incorporated via its p-nitrophenyl active ester. In doing so, Step (11) above was modified to the following 3-step sequence: (a) three washes (10 ml./gram resin) of three minutes each with DMF; (b) addition of 1.0 mmole/gram resin of the p-nitrophenyl ester of N-t-butyloxycarbonylL-asparagine in 10 ml./gram resin of a 1:3 mixture of DMF and methylene chloride followed by mixing for 720 minutes; and (c) three washes (10 ml./gram resin) of three minutes

X-4747A -23each with DMF. Also, Step (20) .above was modified to the use of the p-nitrophenyl ester of N-t-butyloxycarbonylL asparagine in a 3:1 mixture of DMF and methylene chloride followed by mixing for 720 minutes.

The finished peptide-resin was dried in vacuo. The product was hydrolyzed by refluxing for 72 hours in a 1:1 mixture of concentrated hydrochloric acid and dioxane. Amino acid analysis of the resulting product gave the following results, lysine being employed as standard: Asn, 10 1.00; 2Thr, 2.18; Ser, 0.95; Gly, 1.00; Vai, 0.99; 3Phe ,

3.45; 2Lys, 2.00.

Example 3 d-valyl-glycyl-l-cysteinyl-l-lysyll-asparaginyl-l-phenylalanyl-l-phenylALANYL-D-TRYPTOPHYL-L-LYSYL-L-THREONYLL-PHENYLALANYL-L-THREONYL-L-SERYL-L-CYSTEINE

To a mixture of 7.2 ml. of anisole and 7.2 ml. of ethyl mercaptan were added 3.914 grams (at substitution level of 0.155 mmoles/gram) of the protected tetradecapeptide-resin of Example 2. The mixture was cooled in liquid nitrogen, and 80 ml. of liquid hydrogen fluoride were added by distilLation. The resulting mixture was allowed to warm to 0°C. and was stirred for 2 hours. The hydrogen fluoride then was removed by distillation. Ether was added to the remaining mixture, and it was cooled to 0°C. The resulting solid was collected by filtration and washed with ether. The product was dried, and the deprotected tetradecapeptide was extracted from the resin mixture using IM acetic acid and 50% acetic acid. The acetic acid solution then was immediately lyophilized to dryness in the dark.

X-4747A

Β

The resulting slightly yellow solid was suspended in a mixture of 10 ml. of degassed 0.2M acetic acid and 4 ml. of glacial acetic acid. The resulting suspension was heated slightly with 6 ml. of 50% acetic acid until a clean, yellow solution resulted, and the solution was applied to a Sephadex G-25 F column. The chromatographic conditions were: solvent, degassed 0.2M acetic acid; column size, 7.5 x 150 cm.; temperature, 26°C.; flow rate, 16 70 ml./hour; fraction volume, 25.05 ml.

Absorbance at 280 mp of each fraction plotted versus fractionnumber indicated one large broad peak with a following shoulder. UV spectroscopy revealed that the main part of the peak was the product. The fractions which were combined and their effluent volumes are as follows:

Fractions 207-233 (5160-5837 ml., peak = 5515 ml.) This collection of fractions did not include the back side shoulder. UV spectroscopy indicated that 470 mg. of the productwere present, (yield = 46.4%). An Ellman titration of an aliquot indicated a free sulfhydryl content 20 of 95% of theoretical.

Example 4

מ ו

OXIDATION TO D-Val , D-Trp -SOMATOSTATIN

The solution of the reduced D-Val¹, D-Trp⁸somatostatin (677 ml.) from Example 3 was diluted with distilled water to achieve a concentration of 50 pg./ml. Concentrated ammonium hydroxide was added to adjust the pH of. the mixture to 6.7. The solution was stirred at 4°C. in the dark for 64 hours after which an Ellman titration indicated that oxidation was complete.

X-4747A

The mixture was concentrated in vacuo to a volume of 45 ml., and 45 ml. of glacial acetic acid were added. The mixture then was desalted on a Sephadex G-25 F column. The chromatographic conditions were as follows: solvent, degassed 50% acetic acid; column size, 5.0 x 215 cm.; temperature, 26°C.; flow rate, 148 ml./hour; fraction volume, 17.3 ml.

Absorbance at 280 my for each fraction plotted versus fraction number indicated two large peaks. The first 10 peak represented the aggregated forms of the product, and the second peak represented monomeric product. The material represented by the second peak was collected [fractions 116-155 (2000-2685 ml.)]. UV spectroscopy indicated that 279 mg. of product were present in the sample (yield = 59.4%). The solution was lyophilized to dryness in the dark.

The resulting white solid was rechromatographed in two approximately equal portions. The first portion was dissolved in 25 ml. of degassed 50% acetic acid and was 26 absorbed on a Sephadex G-25 F column. Chromatographic conditions were: solvent, degassed 50% acetic acid; column size, 5.0 x 215 cm.; temperature, 26°C.; flow rate, 148 ml./hour; fraction volume, 17.3 ml.

Absorbance at 280 mu for each fraction plotted versus fraction number showed two large peaks. A conservative cut of the second peak was made. Fractions 119-125 (effluent volumes 2128-2256 ml.) were combined. UV spectroscopy indicated that 65.3 mg. of product were present in this sample. The solution was lyophilized to dryness in the 30 dark to obtain the desired product. X-4747A _₂₆_

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The second portion was rechromatographed in the same manner as the first with similar results. The two good . products were combined totalling 126 mg. by UV spectroscopy (45.2% recovery of purified product). The combined product was dissolved in 15 ml. of degassed 0.2M acetic acid and was applied to a Sephadex G-25 F column. Chromatographic conditions were: solvent, degassed 0.2M acetic acid; column size, 5.0 x 150 cm.; temperature, 26°C.; flow rate, 466 ml./hr.; fraction volume, 16.3 ml.

Absorbance at 280 m!1 of each fraction plotted versus fraction number indicated one large peak. UV spectroscopy indicated that the major portion of the peak was excellent product. Fractions 160-180 (2592-2934 ml., peak = 2685 ml.) were combined and were lyophilized to dryness in the dark. UV spectroscopy indicated the presence of 90.4 mg. of product (71.7% recovery).

Optical rotation [a]- -56.1° (1 percent acetic acid).

Amino acid analysis: Vai, 1.0; Gly, 0.97; 2Cys,

1.62; 2Lys, 2.00; Asn, 1.01; 3Phe, 2.87; Trp, 1.02; 2Thr,

1.83; Ser, 0.81.

The above results are expressed as ratios of Lys/2 = 1.0. All values are averages from two 21 hour hydrolyses without scavengers. Tryptophan was determined from UV spectroscopy (as a ratio to Lys/2); serine was not corrected for losses during hydrolysis.

The above product contains minor guantities of impurities. If desired, the product can be further purified

X-4747A by. subjecting it to preparative high pressure liquid chromatography (HPLC).

An alternative method for oxidizing the reduced 18 18

D-Val , D-Trp -somatostatin to D-Val , D-Trp -somatostatin is by treatment with potassium ferricyanide. The oxidation is accomplished in an aqueous solution brought to pH 6.7 as described earlier in this example. An aqueous solution of potassium ferricyanide is added to the mixture to produce a final concentration representing approximately 3.3 times that of the reduced D-Val¹, D-Trp^-somatostatin. The solution is stirred in the dark at room temperature for about two hours. Completion of the oxidation is verified by an Ellman titration.

Example 5

N-t-BUTYLOXYCARBONYL-D-ALANYL-GLYCYL-L-(S-pMETHOXYBENZYL)CYSTEINYL-L- (N^!’-O-CHL0R0BENZYL0XYCARBONYL)LYSYL-L-ASPARAGINYL-L-PHENYLALANYL-LPHENYLALANYL-D-TRYPTOPHYL-L-(N^C-O-CHL0R0-BENZYLOXYCARBONYL)LYSYL-L-(O-BENZYL)THREONYL-LPHENYLALANYL-L-(O-BENZYL)THREONYL-L-(O-BENZYL)SERYL-L-(S-p-METHOXYBENZYL)CYSTEINYL METHYLATED

POLYSTYRENE RESIN

This compound was prepared by using the second portion of the tridecapeptide prepared in Example 2 and coupling N-t-butyloxycarbonyl-D-alanine to it instead of N-t-butyloxycarbonyl-D-valine.

The amino acid analysis of the resulting product after hydrolysis by refluxing for 21 hours in a 1:1 mixture of concentrated hydrochloric acid and dioxane gave the following results, lysine being employed as standard: Asn, 1.16; 2Thr, 2.14; Ser, 1.04; Gly, 1.09; Ala, 1.17; 3Phe, 2.88; 2Lys, 2.0׳).

X-4747A

Example 6

D-ALANYL-GLYCYL-L-CYSTEINYL-L-LYSYLL-ASPARAGINYL-L-PHENYLALANYL-L-PHENYLALANYL-D-TRYPTOPHYL-L-LYSYL-L-THREONYLL-PHENYLALANYL-L-THREONYL-L-SERYL-LCYSTEINE

The title compound was prepared in accordance with the method of Example 3 using 3.772 grams (at substitution level of 0.156 mmole/gram) of the product from Example 5. Purification of the product was accomplished by chromatography on a Sephadex G-25 F column. The chromatographic 10 conditions were: solvent, degassed 0.2M acetic acid;

column size, 7.5 x 150 cm.; temperature, 26°C.; flow rate, 1658 ml./hour; fraction volume, 24.87 ml.

Absorbance at 280 mu of each fraction plotted versus fraction number indicated a large broad peak with a following shoulder. UV spectroscopy showed that the main part of the peak represented the product.

The fractions which were combined and their effluent volumes are as follows:

Fractions 206-230 (5098-5720 ml.)

This collection of fractions did not include the following shoulder. UV spectroscopy indicated a theoretical amount of 403.9 mg. (yield = 41.9%) was present in the sample. An Ellman titration of an aliquot indicated a free sulfhydryl content of 93% of theoretical.

Example 7

מ ו

OXIDATION TO D-Ala , D-Trp -SOMATOSTATIN 1 8

The reduced D-Ala , D-Trp -somatostatin from Example 6 was treated according to the method of Example 4. The solution (622 ml.; theoretically 403.9 mg.) was diluted 30

X-4747A -29with distilled water to achieve a 50 pg./ml. concentration and then was adjusted to pH 6.7 with concentrated ammonium hydroxide. The mixture was stirred at room temperature in the dark for 41 hours. The mixture was concentrated in vacuo to about 40 ml. and then was diluted with 40 ml. of glacial acetic acid. The mixture then was absorbed on a Sephadex G-25 F column. The chromatographic conditions were as follows: solvent, degassed 50% acetic acid; column size, 5.0 x 215 cm.; temperature, 26°C.; flow rate, 151 ml./hour; 10 fraction volume, 17.61 ml.

Absorbance at 280 mp for each fraction plotted versus fraction number indicated two large, broad peaks. The first peak represented aggregated forms of the product, and the second peak represented good monomeric product. The product represented by the second peak [Fractions 113-155 (1971-2728 ml.)] was collected and lyophilized to dryness in the dark. The resulting solid was rechromatographed in approximately two equal portions. The first portion was dissolved in 22 ml. of degassed 50% M acetic acid, and the 20 solution was applied to a Sephadex G-25 F column. The chromatographic conditions were: solvent, degassed 50% acetic acid; column size, 5.0 x 215 cm.; temperature, 26°C.; flow rate, 153 ml./hour; fraction volume, 17.85 ml.

Absorbance at 280 mp of each fraction plotted versus fraction number indicated two large peaks. Λ conservative cut of the second peak was made by combining Fractions 121-127 (effluent volumes of 2142-2267 ml.). UV spectroscopy indicated the presence of 56.7 mg. of product in this sample. The soluti-kn was lyophilized to dryness in the dark to obtain the desired product.

X-4747A -30The second portion was rechromatographed in the same manner as the first. The products were combined (126.3 mg. by UV spectroscopy — 31.3% yield from reduced form). The product was dissolved in 21 ml. of degassed 0.2M acetic acid and was applied to a Sephadex G-25 F column. The chromatographic conditions were: solvent, degassed 0.2M acetic acid; column size, 5.0 x 150 cm.; temperature, 26°C.; flow rate, 449 ml./hour; fraction volume, 15.71 ml.

Absorbance at 280 mp of each fraction plotted versus fraction number indicated one large peak. UV spectroscopy indicated that the major portion of the peak was the product. Fractions 169-188 (2640-2953 ml., peak = 2705 ml.) were combined and lyophilized to dryness in the dark. UV spectroscopy indicated the presence of 85.2 mg. product (67.5% recovery).

Optical rotation [α]^θ - -54.9° (1 percent acetic acid).

Amino acid analysis: Ala, 1.05; Gly, 1.0; 2Cys, 1.58; 2Lys, 2.0; Asn, 1.10; 3Phe, 2.92; Trp, 1.02; 2Thr, 20 1.98; Ser, 0.88.

The above results are expressed as ratios to Lys/2 = 1.0. All values are averages from two 21 hour hydrolyses without scavengers. Tryptophan was determined from UV spectroscopy (as a ratio to Lys/2); serine was not corrected for losses during hydrolysis.

[ g

D-Val־־, D-Trp -somatostatin was tested in dogs for its in vivo inhibition of gastric acid secretion. In six dogs with chronic fistula and Heidenhain pouch, gastric HC1 secretion was induced by infusion of the C-terminal tetra30

X-4747A peptide of gastrin at 0.5 ug./kg.-hr. Each dog served as its own control. After one hour of steady state secretion of HC1, D-Val , D-Trp -somatostatin was infused at 0.15 ug./kg.-hr. for one hour. Collection of gastric acid samples was continued for an additional 1.5 hours at 15 minute intervals. The samples were titrated to pH 7 with an automatic titrator. The maximal inhibitory effect of the 1 8

D-Val , D-Trp -somatostatin was extrapolated against the dose-response curve of somatostatin, and the relative 10 potency of the analog to that of somatostatin is expressed נ r as percent activity. D-Val', D-Trp -somatostatin inhibited steady state acid secretion induced by the C-terminal tetrapeptide of gastrin by 48.22 - 6.45% (standard error of mean). This effect is equivalent to that of 0.175 pg./kg.-hr. of somatostatin. Its activity relative to that of somatostatin thus is 116¾. A more highly purified sample of 18

D-Val , D-Trp -somatostatin administered at doses of 0.200, 0.166, and 0.138 ug./kg.-hr. inhibited steady state acid secretion induced by the C-terminal tetrapeptide of gastrin by 77.63, 71.57, and 67.8%, respectively. This activity relative to that of somatostatin is 302-325%.

8

D-Ala , D-Trp -somatostatin, tested at 0.20 ug./kg.-hr. under the same conditions inhibited steady state acid secretion induced by the C-terminal tetrapeptide of gastrin by 73.61 - 3.66% (standard error of mean). This effect is equivalent to that of 0.550 ug./kg.-hr. of somatostatin. Its activity relative to that of somatostatin thus is 275%.

8 ך

D-Val־, D-Trp -somatostatin and D-Ala¹, D-Trp⁸30 somatostatin also were tested for their action on gut

X-4747A motility in conscious dogs. Three dogs having intralumenal catheters placed in the antrum, duodenum, and pylorus were used. Pressure changes in the gut lumen were recorded on a Visicorder using strain gauges and miniature light beam galvanometers. After a steady state control was established, test compound was infused intravenously over a ten minute period. The test compound initially increased the intralumenal pressure in the pylorus and then decreased it whereas the pressure in the duodenum and the antrum remained depressed throughout the test. The minimum effective dose required to increase the pyloric pressure and to decrease the duodenum and antrum pressures is about 0.05 pg./kg.-10

8 minutes for D-Val , D-Trp -somatostatin and about 0.1 pg./kg.-10 1 8 minutes for D-Ala , D-Trp -somatostatin. This compares to a value for somatostatin itself of 0.125-0.25 pg./kg.-10 minutes.

D-Val¹, D-Trp⁸-somatostatin and D-Ala\ D-Trp⁸somatostatin also were tested for their activity with respect to the release of growth hormone. The procedure 20 which was employed is carried out using normal male SpragueDawley rats weighing 100-120 grams (Laboratory Supply Company, Indianapolis, Indiana). The test is a modification of the method of P. Brazeau, W. Vale, and R. Guilleman, Endocrinology, 94 184 (1974). In this assay, a total of five groups of eight rats each were employed for the testing of each compound. Sodium pentobarbital was administered intraperitoneally to all of the rats to stimulate growth hormone secretion. One group served as the control and received only saline. Two of the groups received somato30

X-4747A statin, one at 2 pg./rat, subcutaneously, and the other at pg./rat, subcutaneously. The other two groups received test compound, one at 10 pg./rat, subcutaneously, and the other at 0.4 pg./rat, subcutaneously. The serum concentration of growth hormone was measured 20 minutes after simultaneous administration of sodium pentobarbital and test compound. The degree of inhibition of serum growth hormone concentration then was determined with respect to the control group, and the relative activities of test compound 10 and of somatostatin itself were compared.

At dose levels of 0.4 pg./rat and 10 pg./rat, 1 8

D-Val , D-Trp -somatostatin inhibited the increase in growth hormone secretion by 14% and by 42% over control, respectively. Somatostatin, at a dose level of 2 pg./rat had no effect on the increase in growth hormone secretion whereas at 50 pg./rat it inhibited the increase in growth hormone secretion by 56% over control.

At dose levels of 0.4 pg./rat and 10 pg./rat, 1 8

D־Ala , D-Trp -somatostatin inhibited the increase in growth 20 hormone secretion by 54% and by.91% over control, respectively. Somatostatin, at dose levels of 2 pg./rat and 50 pg./rat inhibited the increase in growth hormone secretion by 40% and by 87% over control, respectively.

D-Val‘'’, D-Trp®-somatostatin and D-Ala\ D-Trp®somatostatin were tested for.their in vivo activity in inhibiting glucagon and insulin secretion upon stimulation with L-alanine. Normal mongrel dogs of either sex were fasted overnight. Control blood samples were obtained, and then an intravenous infusion of saline, somatostatin, or 30

X-4747A test compound was started. After 30 minutes, L-alanine additionally was administered intravenously for a period of 15 minutes. The infusion of saline, somatostatin, or test compound was continued for 15 minutes after completion of the L-alanine infusion. The infusion of L-alanine caused an abrupt increase in serum concentration of glucagon and insulin which returned to control concentration upon termination of the L-alanine infusion. From the above it was 1 8 determined that the minimal dose of D-Val , D-Trp -somato10 statin for the inhibition of glucagon secretion is 0.04 to 0.11 ug./kg./min. and for the inhibition of insulin secretion is less than 0.004 pg./kg./min. The minimal dose of 18

D-Ala , D-Trp -somatostatin for the inhibition of both glucagon and insulin secretion is less than 0.03 !1g ./kg./min.